Orthopoxviruses (OPVs) are large DNA viruses that can be highly lethal to their natural hosts. Smallpox was produced by the human-specific OPV variola virus (VARV) and was eradicated through vaccination with live vaccinia virus (VACV), a mildly pathogenic OPV. Despite this success, we still know little about the reasons for the high pathogenicity of OPVs in their natural hosts and the mechanisms whereby the smallpox vaccine protects. Studying OPVs is important for human health for several reasons: 1) OPVs are common in many animal species and some of these viruses could jump the species barrier and become human pathogens. 2) There is fear that VARV could be used as a weapon. 3) The vaccine based on live VACV is not safe by current standards and killed VACV does not protect. 4) Since the smallpox vaccine is so effective, understanding how it protects may be valuable to develop vaccines to other viruses, including other large DNA viruses such as herpesviruses. In this project our model will be the mouse OPV ectromelia virus (ECTV) that produces mousepox in susceptible strains of mice. A notable feature of OPVs is their expression of secreted immune response modifiers (IRMs). In Specific Aim 1 we will construct and characterize ECTV mutants that do not express specific IRMs. These mutants will be tested and compared with wild type virus for their growth in tissue culture;their ability to spread and induce pathology in immunodeficient and immunocompetent mice;and the strength and type of humoral and cellular immune response that they induce. Any findings should be applicable to the function of the orthologs of those IRMs in other lethal OPV infections such as smallpox in humans. In Specific Aim 2, we will determine whether specific IRMs and structural proteins exposed to the surface of virions are natural targets of protective immune responses and whether they can be used as vaccines. These experiments are important because they will dissect the mechanisms of protective immunity induced by infection and test a novel approach to non-infectious anti-OPV vaccines.